PH-responsive lipid nanoparticles for mRNA delivery and methods of making same

Abstract

The invention relates to the field of drug delivery, and discloses a pH responsive lipid nanoparticle for mRNA delivery, the lipid nanoparticle comprises the following components: pH responsive lipid molecules with a molar ratio of 30-50%; the invention also discloses a preparation method, which comprises the following steps: dissolving the pH-responsive lipid molecules, cholesterol, phosphatidyl ethanolamine and reduction-responsive lipid molecules in absolute ethyl alcohol to prepare a lipid organic phase, and further comprises the following steps: dissolving the pH-responsive lipid molecules, cholesterol, phosphatidyl ethanolamine and reduction-responsive lipid molecules in absolute ethyl alcohol to prepare the lipid organic phase. Through the dual mechanism design of pH response and reduction response, the delivery efficiency of mRNA is significantly improved, phosphatidyl ethanolamine cooperates with pH response lipid to enhance endosome escape ability and reduce responsive lipid to realize precise cytoplasm release, and meanwhile, through lipid component optimization and preparation process improvement, nanoparticles with controllable particle size and high stability are formed, and the preparation method is simple and easy to implement. The problems of low release efficiency and unstable structure in the prior art are effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery, in particular to pH-responsive lipid nanoparticles for mRNA delivery and a preparation method thereof. Background Art

[0002] In recent years, lipid nanoparticles have been widely used in mRNA vaccines and gene therapy as an efficient nucleic acid delivery system. However, when faced with multiple biological barriers in the delivery path, this technology still has obvious design bottlenecks. First, the existing technology mostly uses cationic lipids such as DODAP to achieve pH response, inducing charge changes through the endosomal acidic environment to destroy the endosomal membrane. Although this mechanism can improve the endosomal escape ability to a certain extent, the design that relies solely on charge interactions is difficult to completely destroy the endosomal membrane structure, resulting in most mRNA being retained in the endosome, weakening its delivery efficiency. Especially when the endosomal environment is complicated (such as local pH fluctuations or changes in membrane lipid composition), the limitations of this design are more prominent. Therefore, the delivery system that relies on a single response mechanism does not perform well in the key link of endosomal escape.

[0003] In the subsequent stage of the delivery pathway, that is, after the mRNA enters the cytoplasm, the release efficiency of existing lipid nanoparticles is also subject to certain limitations. A common strategy is to protect lipid particles through non-responsive PEG modification to prolong their stability in the blood circulation. However, this design exposes significant problems in the second half of the delivery pathway: the rigid PEG protective layer cannot be actively removed in the cytoplasmic environment, hindering the release of mRNA. This not only delays the functional expression of mRNA, but may also cause some mRNA to be inactivated due to degradation by intracytoplasmic nucleases. It can be seen that the existing design has not achieved the optimal balance between stability and release efficiency, and the PEG modification strategy that lacks dynamic responsiveness is obviously unable to meet the needs of complex delivery pathways.

[0004] In addition, the unreasonable optimization of lipid components also makes it difficult for existing delivery systems to balance stability and efficiency. For example, cholesterol, as a key component of particle stability, can enhance the rigidity of the lipid bilayer, but if its proportion is too high, it will inhibit the responsiveness of the particles in the endosomal environment; if it is too low, it will easily lead to the disintegration or aggregation of particles in serum, affecting the delivery efficiency. This imbalance in optimization further leads to uneven particle size distribution, weakening its biodistribution and targeted delivery capabilities. At the same time, the long-term storage stability of existing delivery systems is also obviously insufficient. Especially under freeze-drying conditions, since the particle structure is easily destroyed under physical stress, after re-dissolution, it usually shows an increase in particle size, a decrease in uniformity, and even a loss of delivery function, which seriously restricts the promotion of its practical application.

[0005] In general, existing lipid nanoparticle delivery systems have shown a series of technical deficiencies when facing multiple barriers in the delivery path. These deficiencies are mainly due to the difficulty of a single response mechanism to cover the complex requirements in the delivery path, and the conflict between stability design and functional release has not been effectively resolved. For this reason, there is an urgent need for a lipid nanoparticle delivery system that can achieve precise regulation through a multi-level response mechanism and has both high stability and efficient release capabilities, in order to overcome the limitations of existing technologies and improve the efficiency and practical application value of mRNA delivery. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides pH-responsive lipid nanoparticles for mRNA delivery and a method for preparing the same, which solve the significant deficiencies in the existing mRNA delivery system in terms of endosomal escape efficiency, cytoplasmic release ability, delivery stability, and structural integrity under long-term storage conditions, and are difficult to meet the needs of complex delivery pathways. In particular, the prior art mostly adopts a single response mechanism or rigid modification design, which is unable to dynamically respond to multiple barriers in the delivery pathway, resulting in the problem that the mRNA delivery efficiency and functional expression effect are limited.

[0007] To achieve the above object, the present invention is implemented by the following technical solution: pH-responsive lipid nanoparticles for mRNA delivery, the lipid nanoparticles comprising the following components:

[0008] pH-responsive lipid molecules, with a molar ratio of 30%-50%;

[0009] Helper lipids, molar ratio 30%-50%;

[0010] Reduction-responsive lipids, molar ratio 2%-8%;

[0011] The concentration of the mRNA encapsulated in the lipid nanoparticles is 100-500 μg / mL.

[0012] Preferably, the pH responsive lipid molecule is selected from 1,2-dioleoyl-3-dimethylaminopropane.

[0013] Preferably, the auxiliary lipid comprises cholesterol and phosphatidylethanolamine, wherein the molar ratio of cholesterol is 25%-35%, and the molar ratio of phosphatidylethanolamine is 5%-15%.

[0014] Preferably, the reduction-responsive lipid is selected from DSPE-SS-PEG2000 containing a disulfide bond.

[0015] Preferably, the particle size of the lipid nanoparticles is in the range of 80-150 nm.

[0016] A method for preparing pH-responsive lipid nanoparticles for mRNA delivery, and a method for preparing the pH-responsive lipid nanoparticles for mRNA delivery, comprising the following steps:

[0017] S1, dissolving pH-responsive lipid molecules, cholesterol, phosphatidylethanolamine and reduction-responsive lipid molecules in anhydrous ethanol to prepare a lipid organic phase;

[0018] S2, dissolving the mRNA in PBS buffer to prepare an aqueous phase;

[0019] S3, adding the lipid organic phase to the aqueous phase at a rate of 0.5-2 mL / min through a microinjection pump, while stirring at a speed of 600-1000 rpm to form lipid nanoparticles;

[0020] S4, adding a reduction-responsive lipid molecule solution to the lipid nanoparticle dispersion to form an outer protective structure through self-assembly;

[0021] S5. Purify the nanoparticles to obtain target lipid nanoparticles.

[0022] Preferably, the total lipid concentration of the lipid organic phase in step S1 is 10-20 mg / ml.

[0023] Preferably, the pH value of the mixed solution in step S3 is controlled at 7.0±0.2.

[0024] Preferably, the concentration of the reduction-responsive lipid molecules used in step S4 is 1-5 mg / mL.

[0025] Preferably, in step S5, an ultrafiltration device is used to purify the lipid nanoparticles, and the molecular weight cutoff range of the filter membrane is 10-30 kDa.

[0026] The present invention provides pH-responsive lipid nanoparticles for mRNA delivery and a preparation method thereof, which have the following beneficial effects:

[0027] 1. The present invention adopts a unique dual response mechanism technical solution, combining the dual effects of pH-responsive lipid molecules and reduction-responsive lipids, and successfully realizes precise step-by-step control in the delivery path. By triggering the endosomal escape mechanism in the endosomal acidic environment and further realizing the release of mRNA in the highly reducing environment of the cytoplasm, the delivery efficiency is significantly improved. Compared with the single stimulus-responsive lipid nanoparticle design in the prior art, the present invention can achieve precise regulation of the entire delivery process, overcome the problems of insufficient release efficiency and limited functional expression in the cytoplasm, and greatly optimize the biological effect of mRNA delivery.

[0028] 2. The present invention introduces phosphatidylethanolamine as an auxiliary lipid, utilizes its unique phase transition property of transforming from hexagonal phase I to hexagonal phase II in an acidic environment, and synergizes with the pH-responsive lipid molecule 1,2-dioleoyl-3-dimethylaminopropane to further optimize the endosomal escape efficiency. The membrane fusion property of phosphatidylethanolamine under acidic conditions significantly enhances the endosomal membrane rupture ability, and combines with the charge effect of 1,2-dioleoyl-3-dimethylaminopropane to form a double rupture mechanism. Compared with the delivery method in the prior art that only relies on cationic lipids to induce endosomal membrane disintegration, the present invention is more responsive in the endosomal environment, overcomes the problems of mRNA delivery failure and low delivery efficiency caused by endosomal retention in the prior art, and greatly improves the cytoplasmic release rate of mRNA.

[0029] 3. The present invention adopts a reduction-responsive lipid DSPE-SS-PEG2000 containing disulfide bonds, which can accurately cleave disulfide bonds in a highly reducing cytoplasmic environment and quickly remove the PEG protective layer on the surface of the particles, so that the nanoparticles can achieve effective functional exposure in the cytoplasm. This design effectively avoids the excessive shielding effect brought about by the non-responsive PEG modification strategy in the prior art, ensuring the rapid and complete release of mRNA. Compared with the problem of functional expression being blocked after delivery due to the fixed protective structure of the PEG layer in traditional lipid nanoparticles, the present invention successfully overcomes this important bottleneck in the mRNA release process by using a reduction-responsive strategy, providing a new solution for more efficient gene delivery.

[0030] 4. The present invention has carried out refined optimization on the lipid component ratio and preparation process parameters to form efficient lipid nanoparticles with controllable particle size, uniform surface modification and stable structure. By optimizing the microemulsification process and regulating the lipid molar ratio and outer layer assembly parameters, the present invention can significantly improve the stability of nanoparticles in the blood circulation and effectively avoid their disintegration or degradation due to external environmental interference during the delivery process. Compared with the problems of uneven particle size and easy disintegration of particles in the prior art, the present invention not only prolongs the circulation time of lipid nanoparticles in the body, but also ensures the integrity and delivery efficiency of the structure after delivery to the target cells, providing a reliable structural guarantee for achieving efficient mRNA delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a flow chart of the preparation method. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Please see attached Figure 1 The present invention provides a pH-responsive lipid nanoparticle for mRNA delivery, wherein the lipid nanoparticle comprises the following components:

[0034] pH-responsive lipid molecules, with a molar ratio of 30%-50%;

[0035] Helper lipids, molar ratio 30%-50%;

[0036] Reduction-responsive lipids, molar ratio 2%-8%;

[0037] The mRNA encapsulated in lipid nanoparticles has a concentration of 100-500 μg / mL.

[0038] The pH-responsive lipid molecule is selected from 1,2-dioleoyl-3-dimethylaminopropane.

[0039] 1,2-Dioleoyl-3-dimethylaminopropane is a cationic lipid with a pKa of about 6.5, and is in a neutral state at a neutral pH (7.4), maintaining the stability of the particles.

[0040] In the acidic environment of the endosomal membrane (pH 5.5-6.5), 1,2-dioleoyl-3-dimethylaminopropane dissociates into cations and interacts with the phospholipid bilayer of the endosomal membrane, inducing instability of the endosomal membrane, thereby promoting endosomal rupture and helping mRNA escape into the cytoplasm.

[0041] The auxiliary lipids include cholesterol and phosphatidylethanolamine, wherein the molar ratio of cholesterol is 25%-35%, and the molar ratio of phosphatidylethanolamine is 5%-15%.

[0042] Cholesterol acts as a structural enhancer that can be embedded in the lipid bilayer, improving the membrane stability and fluidity of the particles and preventing early disintegration during blood circulation.

[0043] In an acidic environment, phosphatidylethanolamine transforms from hexagonal phase I (equilibrium stable bilayer structure) to hexagonal phase II (asymmetric non-lamellar structure), further destroying the endosomal membrane, thereby cooperating with 1,2-dioleoyl-3-dimethylaminopropane to achieve endosomal escape.

[0044] The reduction-responsive lipid was selected from DSPE-SS-PEG2000 containing a disulfide bond.

[0045] DSPE-SS-PEG2000 binds the polyethylene glycol protective layer to the particle surface through disulfide bonds, which plays a protective role in blood circulation and prevents nonspecific adsorption and immune clearance.

[0046] When the particles enter the cytoplasm, the high concentration of reducing molecules in the cytoplasmic environment (such as glutathione, with a concentration of about 10mM) reduces the disulfide bonds, removes the PEG protective layer, exposes the inner core of the particles, and promotes the complete release of mRNA.

[0047] The particle size of lipid nanoparticles ranges from 80 to 150 nm.

[0048] Method for preparing pH-responsive lipid nanoparticles for mRNA delivery, method for preparing pH-responsive lipid nanoparticles for mRNA delivery, comprising the following steps:

[0049] S1. Dissolve pH-responsive lipid molecules, cholesterol, phosphatidylethanolamine and reduction-responsive lipid molecules in anhydrous ethanol to prepare a lipid organic phase; the lipid molecules are evenly distributed in the organic phase and form an initial stable intermolecular network through hydrophobic interactions, laying the foundation for subsequent self-assembly

[0050] S2. Dissolve mRNA in PBS buffer to prepare an aqueous phase; PBS provides physiological conditions, and mRNA is negatively charged and can interact with cationic lipid molecules.

[0051] S3. The lipid organic phase is added dropwise to the aqueous phase through a microinjection pump at a rate of 0.5-2 mL / min, while stirring at a speed of 600-1000 rpm to form lipid nanoparticles; the lipid molecules in the organic phase self-assemble in the aqueous phase through hydrophobic and electrostatic interactions to form a lipid bilayer structure to encapsulate mRNA.

[0052] S4. Add reduction-responsive lipid molecule solution to the lipid nanoparticle dispersion to form an outer protective structure through self-assembly; DSPE-SS-PEG2000 embeds its lipid tail into the particle surface to form an outer layer protection, and the PEG part provides a hydrophilic barrier.

[0053] S5. Purify the nanoparticles to obtain target lipid nanoparticles.

[0054] The total lipid concentration of the lipid organic phase in step S1 is 10-20 mg / ml.

[0055] In step S3, the pH value of the mixed solution is controlled at 7.0±0.2.

[0056] The concentration of the reduction-responsive lipid molecule used in step S4 is 1-5 mg / mL.

[0057] In step S5, an ultrafiltration device is used to purify the lipid nanoparticles, and the molecular weight cutoff range of the filter membrane is 10-30 kDa.

[0058] Embodiment 1:

[0059] Preparation steps:

[0060] Preparation of lipid organic phase: In anhydrous ethanol, add 1,2-dioleoyl-3-dimethylaminopropane (molar ratio 40%), cholesterol (molar ratio 30%), phosphatidylethanolamine (molar ratio 10%) and DSPE-SS-PEG2000 (molar ratio 10%), mix well to completely dissolve the lipids, and prepare the lipid organic phase with the concentration controlled at 15 mg / mL.

[0061] Preparation of aqueous phase: mRNA was dissolved in PBS buffer (pH 7.4, containing 100 mM NaCl), and the final concentration was adjusted to 300 μg / mL for later use.

[0062] Formation of lipid nanoparticles: The lipid organic phase was slowly added to the aqueous phase at a rate of 1 mL / min through a microinjection pump, while stirring at 800 rpm on a stirrer. After the addition was completed, stirring was continued for 5 minutes to observe the formation of a uniform nanoparticle dispersion, and the pH was controlled at 7.0 ± 0.2.

[0063] Surface modification: Add DSPE-SS-PEG2000 aqueous solution (2 mg / mL) to the nanoparticle dispersion and stir at room temperature for 30 min to ensure that the PEG protective layer is assembled.

[0064] Purification and particle size control: The particle dispersion was placed in an ultrafiltration device and dialyzed using a 10 kDa MWCO filter membrane to remove unbound lipids and free mRNA. The particle size was controlled by an extruder to obtain lipid nanoparticles with a particle size of 120 nm.

[0065] Embodiment 2:

[0066] Preparation steps:

[0067] Preparation of lipid organic phase: 1,2-dioleoyl-3-dimethylaminopropane (molar ratio 35%), cholesterol (molar ratio 35%), phosphatidylethanolamine (molar ratio 10%) and DSPE-SS-PEG2000 (molar ratio 10%) were dissolved in anhydrous ethanol, and the total lipid concentration was 12 mg / mL.

[0068] Aqueous phase preparation: Dissolve mRNA in PBS buffer and adjust the concentration to 200 μg / mL.

[0069] Assembly of lipid nanoparticles: Use a microinjection pump to add the lipid organic phase to the aqueous phase at a rate of 0.8 mL / min, control the stirring speed at 700 rpm, and continue stirring for 8 minutes after the addition is completed.

[0070] Targeted modification: The folic acid molecules were modified onto the particle surface using the EDC / NHS chemical coupling method with a reaction time of 45 min.

[0071] Purification and freeze-drying: Purification was performed using an ultrafiltration device with a MWCO of 30 kDa. After removing unreacted components, 5% (w / v) trehalose was added as a protective agent and freeze-dried to obtain a stable dry powder.

[0072] Embodiment 3:

[0073] Preparation steps:

[0074] Preparation of lipid organic phase: 1,2-dioleoyl-3-dimethylaminopropane (molar ratio 40%), cholesterol (molar ratio 30%), phosphatidylethanolamine (molar ratio 10%) and DSPE-SS-PEG2000 (molar ratio 10%) were added to anhydrous ethanol, and the lipid concentration was controlled to 20 mg / mL.

[0075] mRNA and aqueous phase configuration: mRNA concentration was adjusted to 400 μg / mL and dissolved in PBS buffer (pH 7.4).

[0076] Nanoparticle formation: The lipid organic phase was added dropwise to the aqueous phase at a rate of 0.5 mL / min through a microinjection pump, and the stirring speed was set to 900 rpm. After stirring for 10 min, uniform nanoparticles were formed.

[0077] Outer layer protection and modification: Use reduction-responsive PEG-modified lipids (DSPE-SS-PEG2000, 3 mg / mL) and control the stirring time to 40 min. Specific peptides are further modified by covalent coupling.

[0078] Particle size optimization: Use an extruder to adjust the particle size to 100-120nm.

[0079] Comparative Example:

[0080] Comparative Example 1: Lipid Nanoparticles Lacking Phosphatidylethanolamine Helper Lipids (Corresponding to Example 1)

[0081] Preparation of lipid organic phase: 1,2-dioleoyl-3-dimethylaminopropane (molar ratio 50%), cholesterol (molar ratio 50%) and DSPE-SS-PEG2000 (molar ratio 10%) were dissolved in anhydrous ethanol, and the lipid concentration was 15 mg / mL.

[0082] Preparation of aqueous phase: Prepare mRNA solution with a concentration of 300 μg / mL and dissolve it in PBS buffer with a pH of 7.4.

[0083] Formation of lipid nanoparticles: The lipid organic phase was added to the aqueous phase at a rate of 1 mL / min through a microinjection pump, and the stirring speed was set to 800 rpm. After the addition was completed, stirring was continued for 5 minutes to form a lipid nanoparticle dispersion, and the pH was controlled at 7.0 ± 0.2.

[0084] Surface modification: Add DSPE-SS-PEG2000 aqueous solution (2 mg / mL) to the dispersion and stir for 30 min to form a surface protection layer.

[0085] Purification and particle size control: Purification was performed using an ultrafiltration device with a MWCO of 10 kDa, and the particle size was finally adjusted to 110-140 nm using an extruder.

[0086] Comparative Example 2: Lipid Nanoparticles without PEG Modification (Corresponding to Example 2)

[0087] Preparation of lipid organic phase: 1,2-dioleoyl-3-dimethylaminopropane (molar ratio 40%), cholesterol (molar ratio 50%) and phosphatidylethanolamine (molar ratio 10%) were dissolved in anhydrous ethanol, and the lipid concentration was 12 mg / mL.

[0088] Preparation of aqueous phase: Prepare mRNA solution with a concentration of 200 μg / mL and dissolve it in PBS buffer.

[0089] Formation of lipid nanoparticles: The lipid organic phase was added dropwise to the aqueous phase at a rate of 0.8 mL / min, the stirring speed was controlled at 700 rpm, and the lipid nanoparticle dispersion was formed after mixing evenly.

[0090] Targeted modification: folic acid was modified onto the particle surface using the EDC / NHS chemical coupling method for 45 min.

[0091] Purification and particle size control: Use an ultrafiltration device with MWCO30kDa for purification, and finally adjust the particle size range to 100-130nm.

[0092] Comparative Example 3: Lipid Ratio Adjustment (Corresponding to Example 3)

[0093] Preparation of lipid organic phase: 1,2-dioleoyl-3-dimethylaminopropane (25% by molar ratio), cholesterol (50% by molar ratio), phosphatidylethanolamine (20% by molar ratio) and DSPE-SS-PEG2000 (5% by molar ratio) were dissolved in anhydrous ethanol, and the lipid concentration was 20 mg / mL.

[0094] Preparation of aqueous phase: Prepare mRNA solution with a concentration of 400 μg / mL and dissolve it in PBS buffer.

[0095] Formation of lipid nanoparticles: The lipid organic phase was added dropwise to the aqueous phase at a rate of 0.5 mL / min by a microinjection pump, and the stirring speed was set to 900 rpm. After the addition was completed, stirring was continued for 10 minutes to form a dispersion.

[0096] Surface protection and modification: Add DSPE-SS-PEG2000 aqueous solution (3 mg / mL) and stir for 40 min to further modify the peptide molecules.

[0097] Purification and particle size control: The particle size range was adjusted to 120-150 nm by an extruder, and the purification conditions were the same as those in Example 3.

[0098] Comparative Example 4: Lipid Nanoparticles without Reduction Response Mechanism (Corresponding to Example 4)

[0099] Preparation of lipid organic phase: 1,2-dioleoyl-3-dimethylaminopropane (30% by molar ratio), cholesterol (50% by molar ratio), phosphatidylethanolamine (15% by molar ratio) and DSPE-PEG2000 (5% by molar ratio, without disulfide bonds) were dissolved in anhydrous ethanol, and the lipid concentration was 18 mg / mL.

[0100] Preparation of aqueous phase: Prepare mRNA solution with a concentration of 250 μg / mL and dissolve it in PBS buffer.

[0101] Nanoparticle assembly: The organic phase was injected into the aqueous phase at a rate of 1.2 mL / min, and the stirring speed was controlled at 750 rpm.

[0102] Freeze-drying protection: After adding 7% (w / v) sucrose solution as a protective agent, freeze-drying was performed.

[0103] Comparative Example 5: Lipid Nanoparticles with Unoptimized Particle Size (Corresponding to Example 3)

[0104] Preparation of lipid organic phase: 1,2-dioleoyl-3-dimethylaminopropane (molar ratio 40%), cholesterol (molar ratio 35%), phosphatidylethanolamine (molar ratio 10%) and DSPE-SS-PEG2000 (molar ratio 10%) were dissolved in anhydrous ethanol, and the lipid concentration was 20 mg / mL.

[0105] Preparation of aqueous phase: Prepare mRNA solution with a concentration of 400 μg / mL and dissolve it in PBS buffer.

[0106] Nanoparticle assembly: The organic phase was added dropwise to the aqueous phase at a rate of 0.5 mL / min, and the stirring speed was controlled at 900 rpm to form a lipid nanoparticle dispersion.

[0107] Surface protection and modification: DSPE-SS-PEG2000 was used for surface modification, and the modification time was 40 min.

[0108] Purification: Purification was performed using an ultrafiltration device with MWCO 30 kDa, but without extruder particle size control.

[0109] Experiment 1: mRNA delivery efficiency comparison experiment

[0110] Experimental description:

[0111] Cell preparation: HEK293 cells were seeded in 96-well plates, 200 μL of complete medium was added to each well, and the cell density was controlled at 1×10 5 The cells were cultured in a 37°C, 5% CO2 incubator for 24 h, and the experiment was started when the cell confluence reached 80%.

[0112] Nanoparticle treatment: Lipid nanoparticles prepared in Examples 1, 2, 3 and Comparative Examples 1, 2, 3 were used to deliver EGFP encoding mRNA (concentration 200 ng / mL). Each nanoparticle was immediately added to the cell culture medium (200 μL / well) after preparation, and three replicate wells were set for each treatment. The blank control group was only added with culture medium.

[0113] Culture and detection: The treated cells were incubated in a 37°C, 5% CO2 incubator for 6 h, then washed three times with PBS buffer to remove free particles and unabsorbed mRNA. After adding 200 μL of fresh culture medium, the cells were cultured for another 48 h.

[0114] Fluorescence detection: At 48h, the fluorescence intensity of EGFP expression was observed using a fluorescence microscope and the image was recorded. Then, flow cytometry was used to analyze the proportion of EGFP-positive cells in each group after treatment (EGFP-positive cells refer to the number of cells with significantly higher fluorescence intensity than the blank control).

[0115] Experimental data:

[0116] Table 1: Experimental data of EGFP positive cell ratio

[0117]

[0118]

[0119] Experimental summary:

[0120] The efficiency of lipid nanoparticles in delivering mRNA is obviously affected by their structural and functional design. It can be seen from the experimental results that the mRNA delivery efficiency of the embodiment is significantly higher than that of the corresponding comparative example. In particular, after the addition of phosphatidylethanolamine auxiliary lipids in Example 1, the proportion of positive cells was significantly improved compared with Comparative Example 1. This shows that the phase change properties of phosphatidylethanolamine promote endosomal membrane rupture in an acidic environment, thereby enhancing the endosomal escape ability. Compared with Comparative Example 1, which relies solely on the single mechanism of 1,2-dioleoyl-3-dimethylaminopropane, the synergistic effect of phosphatidylethanolamine and 1,2-dioleoyl-3-dimethylaminopropane provides a dual endosomal rupture mechanism, solving the problem of endosomal retention.

[0121] The introduction of PEG modification also has a significant effect on the delivery efficiency. Example 2 shows a higher mRNA expression efficiency than Comparative Example 2, which can be attributed to the presence of the PEG protective layer, which prolongs the stability of the nanoparticles in the blood circulation and prevents the degradation or nonspecific adsorption of the particles outside the cells. The lack of PEG modification in Comparative Example 2 can effectively deliver some mRNA, but in a complex culture environment, the integrity of the nanoparticles is significantly reduced, which ultimately affects the delivery efficiency. This further verifies the importance of the reduction response mechanism. The PEG protective layer will not interfere with the mRNA release after being removed by reducing conditions in the cytoplasmic environment. The designed multi-level response system has practical advantages.

[0122] In addition, the optimization of the lipid component ratio obviously also plays a key role. The experimental results of Example 3 show that a reasonable lipid ratio (the coordinated ratio of 1,2-dioleoyl-3-dimethylaminopropane to phosphatidylethanolamine and cholesterol) can strike a balance between stability and endosomal escape ability. Compared with Comparative Example 3, Example 3 provides a more stable structure while maintaining a high delivery efficiency. This proves that in the design of lipid nanoparticles, the lipid component ratio not only determines the basic structural characteristics of the particles, but also directly affects the delivery effect of mRNA. The adjustment of the ratio in the present invention exposes its inadequate problem in the delivery path, and the optimized design of the embodiment solves this bottleneck.

[0123] Experiment 2: Testing the endosomal escape ability of lipid nanoparticles

[0124] Experimental description:

[0125] Cell preparation: HEK293 cells were seeded in glass-bottom culture dishes. 1 mL of complete culture medium was added to each dish and the cell density was adjusted to 5 × 10 5 The cells were cultured at 37°C in a 5% CO2 environment for 24 h until the cells attached and grew to 80% confluence.

[0126] Endosome / lysosome labeling: LysoTrackerGreen (1 μM, dissolved in serum-free medium) was used to label the endosomes and lysosomes in the cells. The staining solution was added to the cell culture dish and incubated at 37°C for 30 minutes, and then the cells were washed 3 times with serum-free medium, each time for 5 minutes.

[0127] Nanoparticle treatment: Using the lipid nanoparticles prepared in Examples 1 and 2 and Comparative Examples 1 and 2, the nanoparticles containing Cy5-labeled mRNA (at a concentration of 200 ng / mL) were added to the labeled cell culture dish and incubated at 37° C. for 2 h. Three replicates were set for each treatment.

[0128] Fluorescence imaging: After incubation, cells were washed three times with PBS and replaced with fresh culture medium. Confocal laser scanning microscopy was used to observe the distribution of Cy5 signal (red) and LysoTracker signal (green), and the fluorescence images of each group were recorded.

[0129] Fluorescence colocalization analysis: The colocalization coefficient of red and green fluorescence was analyzed using ImageJ software. A decrease in the colocalization coefficient indicates an enhanced ability of the nanoparticles to escape from endosomes / lysosomes.

[0130] Experimental data:

[0131] Table 2: Experimental data on the colocalization coefficient of lipid nanoparticles' endosomal escape ability

[0132]

[0133] Experimental summary:

[0134] From the experimental results, Example 1 and Comparative Example 1 show significant differences in endosomal escape ability. The addition of phosphatidylethanolamine is the key. Phosphatidylethanolamine undergoes a phase transition in an acidic environment, from hexagonal phase I to hexagonal phase II, resulting in local rupture of the endosomal membrane structure. In contrast, Comparative Example 1 only relies on the pH responsiveness of 1,2-dioleoyl-3-dimethylaminopropane. This single mechanism cannot fully perturb the endosomal membrane, so the colocalization coefficient is higher, indicating that more nanoparticles are still retained in the endosome. The synergistic effect of phosphatidylethanolamine and 1,2-dioleoyl-3-dimethylaminopropane shows practical advantages in the endosomal escape process, which is the basis for Example 1 to achieve efficient delivery.

[0135] The effect of PEG modification is also reflected in the comparison between Example 2 and Comparative Example 2. In Example 2, the reduction-responsive PEG modification layer can be removed in the cytoplasmic environment, avoiding the interference of the PEG layer on the endosomal escape. In Comparative Example 2, the non-responsive PEG modification was used, and although the stability of the particles was improved, the presence of the PEG layer in the endosomal environment hindered the interaction between the particles and the endosomal membrane. The difference in the colocalization coefficient shows that the design of removing PEG protection is particularly important for achieving efficient delivery to the cytoplasm.

[0136] From the perspective of the overall efficiency of endosomal escape, the combination of pH response and phase change lipids, as well as the precise stripping of the reduction response layer, jointly construct a hierarchical delivery mechanism. Each set of data in the experiment can be traced back to a specific molecular point of action. Examples 1 and 2 not only reflect the rationality of the design ideas, but also emphasize the synergistic needs of endosomal membrane rupture and precise release, while the comparative examples reflect the decreased efficiency caused by the lack of these mechanisms. These results further prove that the optimized lipid design of the present invention can effectively solve the endosomal retention problem in the background technology.

[0137] Experiment 3: Assessment of cytoplasmic release capacity

[0138] Experimental description:

[0139] Cell preparation: HEK293 cells were seeded in 24-well plates, and 500 μL of complete medium was added to each well. The density was controlled at 2×10 5 cells / well, culture for 24 h, and start the experiment when the cell confluence reaches 70%-80%.

[0140] Lipid nanoparticle delivery: The lipid nanoparticles of Example 2 and Comparative Example 4 were used to load Cy5-labeled mRNA (concentration 300 ng / mL). After the nanoparticle dispersion was prepared, 100 μL was added to each well, and the final mRNA concentration was 50 ng / mL. A blank control group was set up with only culture medium added, without nanoparticle treatment.

[0141] Culture and treatment: The culture plates were placed in a 37°C, 5% CO2 incubator for 4 h. After incubation, the cells were washed three times with PBS buffer to remove free nanoparticles. Fresh culture medium was added and incubated for another 2 h.

[0142] Cytoplasmic signal detection: Fluorescence microscopy observation: Use a fluorescence microscope to directly observe the Cy5 signal (red) in the cells and record the distribution of mRNA in the cytoplasm.

[0143] Fluorescence spectrophotometer detection: Collect cells, lyse and detect the fluorescence intensity of mRNA in the cytoplasm. Add cytoplasmic lysate to a 96-well plate, 100 μL per well, and measure the fluorescence intensity using a fluorescence spectrophotometer (excitation wavelength 649 nm, emission wavelength 670 nm).

[0144] Experimental data:

[0145] Table 3: Experimental data of fluorescence intensity of Cy5-labeled mRNA in cytoplasm

[0146]

[0147]

[0148] Experimental summary:

[0149] The experiment clearly shows that the cytoplasmic release ability of Example 2 is significantly better than that of Comparative Example 4. The design of reduction-responsive DSPE-SS-PEG2000 plays a crucial role in cytoplasmic release. In the highly reducing environment of the cytoplasm, the disulfide bonds of DSPE-SS-PEG2000 are broken, and the PEG protective layer is quickly detached, exposing the core structure of the lipid nanoparticles, thereby promoting the release of mRNA. Although the non-reduction-responsive PEG used in Comparative Example 4 provides certain protection during the delivery process, its non-peelable characteristics ultimately hinder the release of mRNA, and the cytoplasmic fluorescence signal is significantly reduced. This design difference directly reveals the practical value of the reduction-responsive mechanism.

[0150] The release of mRNA in cells is not a simple physical diffusion process, but depends on multi-level structural responses. In Example 2, lipid nanoparticles can accurately release payloads from the cytoplasm, which is the result of the combined effect of multiple designs. In particular, the removability of the reduction response layer completes structural changes through biological specific stimulation in the cytoplasmic environment, rather than mechanical disintegration. This design obviously effectively solves the problem of low cytoplasmic release efficiency in the background technology, which is crucial to the improvement of delivery efficiency.

[0151] It can also be seen from the experiment that the fluorescence signal of the blank control group can be basically ignored, and although comparative example 4 shows a certain ability in delivering mRNA, the release efficiency is still significantly limited compared with example 2. There is no doubt that the design core of example 2 lies in the realization of dynamic responsiveness in the delivery path. This functional design can adapt to the specific conditions of the cytoplasmic environment, provides a precise release mechanism for mRNA delivery, and significantly enhances the activity of functional molecules in cells.

[0152] Experiment 4: Stability test of nanoparticles

[0153] Experimental description:

[0154] Serum stability test:

[0155] Sample preparation: The lipid nanoparticles prepared in Examples 3 and 4 and Comparative Examples 3 and 5 (final concentration of 200 μg / mL) were added to PBS solution containing 50% fetal bovine serum (FBS) to simulate the blood circulation environment in vivo.

[0156] Culture conditions: Place the sample in a 37°C water bath, take samples every 2 hours, and record the changes in particle size at 0 hours, 2 hours, 4 hours, and 8 hours.

[0157] Detection method: Dynamic light scattering (DLS) was used to measure the particle size distribution to determine the stability of the nanoparticles.

[0158] Freeze-dried stability test:

[0159] Sample preparation: The lipid nanoparticles of Example 4 and Comparative Example 4 were taken, 7% (w / v) sucrose protective agent was added, and then freeze-dried and stored at 4°C.

[0160] Redissolution test: Take samples on the 0th and 30th day after freeze-drying and reconstitute with PBS. Detect the change in particle size and zeta potential after reconstitution to analyze whether the particle structure is stable.

[0161] Detection method: DLS was used to determine the particle size, and zeta potential analyzer was used to determine the surface charge.

[0162] Experimental data:

[0163] Table 4: Particle size stability of lipid nanoparticles in serum and lyophilized conditions

[0164]

[0165]

[0166] Experimental summary:

[0167] The experimental results show that the particle size of Example 3 under serum conditions changes relatively slowly, with the average particle size gradually increasing from the initial 123.7 nm to 167.7 nm after 8 hours, while the particle size of Comparative Example 3 increases dramatically, reaching 310.0 nm at 8 hours. This clearly shows that the reasonably optimized lipid ratio in Example 3 provides good structural stability of the particles, and the synergistic effect of cholesterol and phosphatidylethanolamine enhances the stability of the lipid bilayer, thereby slowing down the rate of protein adsorption and particle aggregation in the serum environment. However, the lipid ratio in Comparative Example 3 that is not fully optimized causes the particles to disintegrate or aggregate rapidly in serum, and the final stability is greatly reduced.

[0168] The freeze-drying stability experiment further reveals the importance of reduction response design to the maintenance of particle structure. After adding sucrose as a cryoprotectant in Example 4, the reconstituted particle size only changed slightly within 30 days (increased from 125.0nm to 130.7nm), showing excellent freeze-drying stability. The non-responsive PEG-modified particles in Comparative Example 4, the particle size after reconstitution increased significantly to 195.0nm at 30d, reflecting the lack of particle structural integrity. This result can be traced back to the limitations of non-responsive PEG modification, whose rigid structure is more susceptible to stress damage during freeze-drying and reconstitution, causing the particles to lose uniformity.

[0169] Based on the serum stability and freeze-drying experimental data, the advantage of the embodiment is that the structural design and the optimization of process parameters jointly achieve high stability of the delivery particles. The dynamic response design is particularly critical. DSPE-SS-PEG2000 provides short-term protection in the serum environment and achieves precise stripping under reducing conditions, avoiding the interference of rigid modification on the delivery process. This hierarchical and dynamically regulated design solves the problem of instability of traditional lipid nanoparticles in complex in vitro and in vivo environments, laying the foundation for the practical application of mRNA drug delivery.

[0170] Experiment 5: In vivo verification of mRNA delivery efficiency and functional expression

[0171] Experimental description:

[0172] Experimental animals: Healthy 8-week-old BALB / c mice (half male and half female) were selected, 5 in each group, and randomly divided into example groups (Examples 1, 2, and 3) and comparative example groups (Comparative Examples 1, 2, and 3). Each group was injected with lipid nanoparticles through the tail vein, and the blank control group was injected with PBS solution.

[0173] Preparation of lipid nanoparticles: Each group of lipid nanoparticles was loaded with EGFP encoding mRNA, and the delivery dose was set to 0.5 mg / kg. After the lipid particles were prepared, dynamic light scattering (DLS) was used to confirm that their particle size was in the range of 100-150 nm to ensure the uniformity of the delivered particles.

[0174] Injection and tissue collection:

[0175] Each mouse was injected with lipid nanoparticles via the tail vein and sacrificed under anesthesia 24 hours later.

[0176] The liver, spleen and lung tissues were taken separately, weighed, washed three times with PBS to remove blood, and immediately stored in liquid nitrogen.

[0177] Fluorescent protein expression detection:

[0178] Fluorescence imaging: The frozen tissues were sliced ​​(thickness 10 μm), and the distribution of EGFP was observed using a fluorescence microscope and the fluorescence images were recorded.

[0179] Western Blut: After extracting tissue proteins, Western Blut was used to analyze the expression of EGFP protein, using β-actin as an internal reference.

[0180] Quantification of tissue distribution: The EGFP fluorescence signal in the tissue was quantitatively analyzed using a fluorescence spectrophotometer. After tissue lysis, the supernatant was taken for measurement, with an excitation wavelength of 488 nm and an emission wavelength of 510 nm.

[0181] Experimental data:

[0182] Table 5: Experimental data of EGFP fluorescence intensity in various tissues

[0183]

[0184] Experimental summary:

[0185] The experimental results clearly reveal the advantages of the lipid nanoparticles of the present invention in in vivo delivery efficiency. Compared with Comparative Example 1, the EGFP fluorescence intensity of Example 1 is significantly improved, especially in the liver, where the delivery effect is most significant. This is mainly due to the phase transition characteristics of phosphatidylethanolamine. In liver cells, the acidic endosomal environment triggers the transition of phosphatidylethanolamine from a bilayer structure to a hexagonal phase II, directly destroying the endosomal membrane. This mechanism obviously enhances the endosomal escape ability of mRNA. However, due to the lack of phosphatidylethanolamine, Comparative Example 1 only relies on the pH responsiveness of DODAP, which is not enough to destroy the endosomal membrane, resulting in mRNA retention and limited functional expression.

[0186] The contribution of PEG modification is also particularly evident in Example 2 and Comparative Example 2. The DSPE-SS-PEG2000 in Example 2 breaks disulfide bonds due to reducing conditions in the cytoplasmic environment, and the core particles are released after the PEG layer is peeled off, achieving efficient mRNA release. In contrast, the non-responsive PEG layer that cannot be peeled off in Comparative Example 2 hinders the release of mRNA from the particles. Even if it is delivered to the tissue, the functional expression of mRNA is still significantly limited. This dynamic response design is obviously the key point to solve the multi-layer barrier in the complex delivery path.

[0187] The optimization of lipid components is also reflected in the results of Example 3. The rationally distributed cholesterol ratio enhances the membrane stability of the particles, while the addition of phosphatidylethanolamine enhances the ability of endosome rupture. The unbalanced lipid ratio in Comparative Example 3 leads to insufficient stability of the particles in vivo, affecting the delivery effect. It can be seen from the difference in the distribution of fluorescent signals that the optimized lipid nanoparticles can achieve a balance between stability and delivery efficiency, solving the problem that stability and delivery efficiency are difficult to achieve in the background technology. Ultimately, the optimized design of the embodiment shows significant advantages in the efficiency and functional expression of mRNA delivery in vivo through synergistic effects.

[0188] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. pH-responsive lipid nanoparticles for mRNA delivery, characterized in that The lipid nanoparticles include the following components: pH-responsive lipid molecules, with a molar ratio of 30%-50%; Helper lipids, molar ratio 30%-50%; Reduction-responsive lipids, molar ratio 2%-8%; The concentration of the mRNA encapsulated in the lipid nanoparticles is 100-500 μg / mL.

2. The pH-responsive lipid nanoparticle for mRNA delivery according to claim 1, characterized in that The pH responsive lipid molecule is selected from 1,2-dioleoyl-3-dimethylaminopropane.

3. The pH-responsive lipid nanoparticle for mRNA delivery according to claim 1, characterized in that The auxiliary lipids include cholesterol and phosphatidylethanolamine, wherein the molar ratio of cholesterol is 25%-35%, and the molar ratio of phosphatidylethanolamine is 5%-15%.

4. The pH-responsive lipid nanoparticle for mRNA delivery according to claim 1, characterized in that The reduction-responsive lipid is selected from DSPE-SS-PEG2000 containing a disulfide bond.

5. The pH-responsive lipid nanoparticle for mRNA delivery according to claim 1, characterized in that The particle size of the lipid nanoparticles ranges from 80 to 150 nm.

6. A method for preparing pH-responsive lipid nanoparticles for mRNA delivery, characterized in that: The method for preparing the pH-responsive lipid nanoparticles for mRNA delivery according to any one of claims 1 to 5 comprises the following steps: S1, dissolving pH-responsive lipid molecules, cholesterol, phosphatidylethanolamine and reduction-responsive lipid molecules in anhydrous ethanol to prepare a lipid organic phase; S2, dissolving the mRNA in PBS buffer to prepare an aqueous phase; S3, adding the lipid organic phase to the aqueous phase at a rate of 0.5-2 mL / min through a microinjection pump, while stirring at a speed of 600-1000 rpm to form lipid nanoparticles; S4, adding a reduction-responsive lipid molecule solution to the lipid nanoparticle dispersion to form an outer protective structure through self-assembly; S5. Purify the nanoparticles to obtain target lipid nanoparticles.

7. The method for preparing pH-responsive lipid nanoparticles for mRNA delivery according to claim 6, characterized in that: The total lipid concentration of the lipid organic phase in step S1 is 10-20 mg / ml.

8. The method for preparing pH-responsive lipid nanoparticles for mRNA delivery according to claim 6, characterized in that: The pH value of the mixed solution in step S3 is controlled at 7.0±0.

2.

9. The method for preparing pH-responsive lipid nanoparticles for mRNA delivery according to claim 6, characterized in that: The concentration of the reduction-responsive lipid molecules used in step S4 is 1-5 mg / mL.

10. The method for preparing pH-responsive lipid nanoparticles for mRNA delivery according to claim 6, characterized in that: In the step S5, an ultrafiltration device is used to purify the lipid nanoparticles, and the molecular weight cutoff range of the filter membrane is 10-30 kDa.

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